Buffy coat separator float system and method

ABSTRACT

A tube and float system for use in separation and axial expansion of the buffy coat includes a transparent or semi-transparent, flexible sample tube and a rigid separator float having a specific gravity intermediate that of red blood cells and plasma. The float includes a main body portion of reduced diameter to provide a clearance gap between the inner wall of the sample tube and the float. One or more protrusions on the main body portion serve to support the flexible tube. During centrifugation, the centrifugal force causes the diameter of the flexible tube to expand and permit density-based axial movement of the float in the tube. The float further includes a pressure relief system to alleviate pressure build up in the trapped red blood cell blood fraction below the float, thereby preventing red blood cells from being forced into the annular gap containing the buffy coat layers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/029,289, filed Feb. 11, 2008, which is a continuation of U.S. patentapplication Ser. No. 11/609,186, filed Dec. 11, 2006, now U.S. Pat. No.7,358,095, which in turn is a continuation of U.S. patent applicationSer. No. 10/263,974, filed Oct. 3, 2002, now U.S. Pat. No. 7,074,577,all of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to density-based fluidseparation and, in particular, to an improved sample tube and floatdesign for the separation and axial expansion of constituent fluidcomponents layered by centrifugation, and a method employing the same.The present invention finds particular application in blood separationand axial expansion of the buffy coat layers, and will be described withparticular reference thereto. However, it will be recognized that thepresent invention is also amenable to other like applications.

BACKGROUND OF THE INVENTION

Quantitative Buffy Coat (QBC) analysis is routinely performed inclinical laboratories for the evaluation of whole blood. QBC analysistechniques generally employ centrifugation of capillary tubes containinganticoagulated whole blood, to separate the blood into six distinctlayers: (1) packed red cells, (2) reticulocytes, (3) granulocytes, (4)lymphocytes/monocytes, (5) platelets, and (6) plasma. Based onexamination of the tube, the length or height of essentially each layeris determined and converted into a cell count, thus allowingquantitative measurement of each layer. The length can be measured witha manual reading device, i.e., a magnification eyepiece and a manualpointing device, or photometrically by an automated optical scanningdevice that finds the layers by measuring light transmittance andfluorescence along the length of the tube. A series of commonly used QBCinstruments are manufactured by Becton-Dickinson and Company of FranklinLakes, N.J.

Since the buffy coat layers are very small, the buffy coat is oftenexpanded in the tube for more accurate visual or optical measurement byplacing a plastic cylinder, or float, into the tube. The float has adensity which is less than that of red blood cells (1.090 g/ml) andgreater than that of plasma (1.028 g/ml) and occupies nearly all of thecross-sectional area of the tube. The volume-occupying float, therefore,generally rests on the packed red blood cell layer and greatly expandsthe axial length of the buffy coat layers in the tube for analysis.

There exists a need in the art for an improved sample tube and floatsystem and method for separating blood and/or identifying circulatingcancer and/or other rare cells, organisms or particulates or objects(i.e., stem cells, cell fragments, virally-infected cells, trypanosomes,etc.) in the buffy coat or other layers in a blood sample. However, thenumber of cells expected to be typically present in the buffy coat isvery low relative to the volume of blood, for example, in the range ofabout 1-100 cells per millimeter of blood, thus making the measurementdifficult, particularly with the very small sample sizes employed withthe conventional QBC capillary tubes and floats.

The present invention contemplates a new and improved blood separationassembly and method that overcome the above-referenced problems andothers.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, an apparatus for separationand analysis of a target analyte in sample of anticoagulated whole bloodis provided. The apparatus includes a transparent or semi-transparent,flexible tube for holding the sample and an elongate, rigid,volume-occupying float having a specific gravity intermediate that ofred blood cells and plasma. The float comprises a main body portionspacedly surrounded by the inner peripheral surface of the sidewall ofthe tube to form an annular volume therebetween. One or more supportmembers protrude from the main body portion to engage and support thesidewall. An internal passage extends axially through the main bodyportion of the float. The sidewall of the tube is resiliently radiallyexpandable in response to centrifugal force so as to permit axialmovement of the float in the tube and fluid flow therearound duringcentrifugation. The internal passage is present to prevent excessive,disruptive fluid flow through the separated buffy coat layers caused bythe collapse of the outer wall of the sample tube to form the analysisarea during the deceleration period of centrifugation.

In a second aspect, a method of separating and axially expanding buffycoat constituents in a blood sample comprises introducing the bloodsample into a flexible sample tube having an elongate side wall with aninner peripheral surface. An elongate, rigid volume-occupying float,which has a specific gravity intermediate that of red blood cells andplasma, is introduced into the flexible sample tube. The float comprisesa main body portion spacedly surrounded by the inner peripheral surfaceof the sidewall of the tube to form an annular volume therebetween. Oneor more support members protrude from the main body portion of the floatto engage and support the sidewall and an internal passage extendsaxially through the main body portion. The sample is centrifuged toeffect a density-based separation of the blood sample into discretelayers at a rotational speed that causes a resilient radial expansion ofthe tube sidewall to a diameter that is sufficiently large to permitaxial movement of the float in the tube. The float moves into axialalignment with at least the buffy coat layers of the blood sample inresponse to the centrifugal force and, thereafter, the rotational speedis reduced to cause the tube sidewall inner surface to capture thefloat.

In a third aspect, a volume occupying separator float is provided. Thefloat is adapted for use with an associated sample tube and comprises arigid main body portion and one or more rigid tube support membersextending radially outwardly from the main body portion. The tubesupport members are sized to engage an inner wall of the sample tube andconfigured to maintain a clearance gap between the main body portion andthe inner wall of the sample tube. The float further comprises means foralleviating excessive flow through the expanded cell layers present inthe clearance gap during centrifugation.

In a fourth aspect, a method for detecting circulating epithelial cancercells in an anticoagulated whole blood sample comprises combining theblood sample with one or more epithelial cell epitope-specific labelingagents so as to differentiate epithelial cancer cells from other cellsin the blood sample. The blood sample is introduced into a transparentsample tube comprising a flexible sidewall having an inner peripheralsurface and a volume-occupying separator float is inserted into thesample tube. The separator float comprises a rigid main body portionhaving a cross-sectional diameter less than an inner diameter of thesample tube and one or more rigid tube support members extendingradially outwardly from the main body portion. The support members aresized to engage an inner wall of the sample tube and are configured tomaintain a clearance gap between the main body portion and the innerwall. The separator float further comprises a pressure relief system forautomatically relieving any pressure differential across opposite axialends of the float as a result of centrifuging. The blood sample andseparator float are centrifuged to effect centrifugally motivatedlocalization of any epithelial cancer cells present in the blood samplewithin the clearance gap. After centrifuging, the blood sample isexamined for the presence of epithelial cancer cells contained in theclearance gap, i.e., the analysis area.

In a still additional aspect, the compressibility and/or rigidity of theflexible sample tube and rigid float can be reversed. In this aspect,the float is designed to shrink in diameter at the higher pressures andmoves freely within a rigid, or optionally, semi-rigid tube. The use ofa compressible float allows for usage of transparent glass tubes, which,in some instances, exhibit enhanced optical properties over polymerictubes. Furthermore, this aspect generally reduces the tolerancerequirements for the glass tubes (since the float would expand upagainst the tube wall after the pressure decreases), and a full range offloat designs is possible.

In another aspect, the step of centrifugation is not required. In suchan aspect, the application of pressure alone to the inside of the tube,or simply the expansion of the tube (or the compression of the float) isrequired. For example, such pressure can be produced through the use ofa vacuum source on the outside of the tube. Such an application alsoallows for the top of the sample tube to be kept open and easilyaccessible. Additionally, the use of a vacuum source may be easier toimplement in some situations than the application of a centrifugalforce.

Additionally, any method of tubular expansion/contraction (or floatcompression) such as mechanical, electrical, magnetic, etc., can beimplemented. Once the tube is expanded (or the float is compressed), thefloat will move to the proper location due to buoyancy forces created bythe density variations within the sample.

In a further aspect, the float comprises a part of a flexible collectiontube system or assembly. In this aspect, it is not necessary to transferthe sample from a collection container to an analysis tube. The blood orsample fluid can be collected immediately and then tested. Such a systemis somewhat faster, and also safer from a biohazard standpoint. Forexample, this system is desirable in very contagious situations (i.e.Ebola virus, HIV, etc.) where any type of exposure of the blood must beminimized.

One advantage of the present invention is found in a blood separatingapparatus that can separate the entire buffy coat of a relatively largeblood sample from the rest of the blood volume.

Another advantage of the invention resides in the fact that the buffycoat layers can be made available for visualization or imaging in onesimple operation, i.e., centrifugation.

Still another advantage of the invention resides in enhanced buffy coatseparation, retention, and, if desired, removal from the sample tube forfurther processing.

Another advantage of the invention resides in that reducedcentrifugation speeds can be used to spin down the blood sample, therebyreducing possible tube failures.

Still another advantage is found in that the tube can be supported forimproved imaging of the sample, and a more repeatable depth for imagingmay be provided.

Still further advantages of the present invention reside in itsrelatively simple construction, ease of manufacture, and low cost.

Another advantage resides in that pressure beneath the float isautomatically alleviated, thereby reducing contamination of theseparated buffy coat by intruding red blood cells.

Still further advantages and benefits of the present invention willbecome apparent to those of ordinary skill in the art upon reading andunderstanding the following detailed description of the preferredembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may take form in various components and arrangements ofcomponents, and in various steps and arrangements of steps. Thedrawings, in which like reference numerals denote like componentsthroughout the several views, are only for purposes of illustratingpreferred embodiments and are not to be construed as limiting theinvention.

FIG. 1 is a sectional view of a sample tube containing a generallyspool-shaped separator float with a central bore according to anexemplary embodiment of the invention.

FIG. 2 is an elevational view of a separator float having generallyconical ends according to another exemplary embodiment of the invention.

FIG. 3 is an elevational view of a separator float having axiallyspaced-apart ribs according to a further exemplary embodiment of theinvention.

FIG. 4 is a perspective view of a separator float having axiallyextending ridges or splines according to yet another exemplaryembodiment.

FIG. 5 is an exploded perspective view of a two-piece separator floataccording to still another exemplary embodiment of the invention.

FIGS. 6-12 are side sectional views of additional exemplary two-piecefloat embodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to the drawings, wherein the showings are for purposes ofillustrating the preferred embodiments of the invention only and not forlimiting the same, FIG. 1 shows a blood separation tube and floatassembly 100, including a sample tube 130 having a separator float orbobber 110 of the invention therein.

The sample tube 130 is generally cylindrical in the depicted embodiment,although tubes having polygonal and other geometrical cross-sectionalshapes are also contemplated. The sample tube 130 includes a first,closed end 132 and a second open end 134 receiving a stopper or cap 140.Other closure means are also contemplated, such as parafilm or the like.In alternative embodiments, not shown, the sample tube may be open ateach end, with each end receiving an appropriate closure device.

Although the tube is depicted as generally cylindrical, the tube 130 maybe minimally tapered, slightly enlarging toward the open end 134,particularly when manufactured by an injection molding process. Thistaper or draft angle is generally desirable for ease of removal of thetube from the injection-molding tool.

The tube 130 is formed of a transparent or semi-transparent material andthe sidewall 136 of the tube 130 is sufficiently flexible or deformablesuch that it expands in the radial direction during centrifugation,e.g., due to the resultant hydrostatic pressure of the sample undercentrifugal load. As the centrifugal force is removed, the tube sidewall136 substantially returns to its original size and shape.

The tube may be formed of any transparent or semi-transparent, flexiblematerial (organic and inorganic), such as polystyrene, polycarbonate,styrene-butadiene-styrene (“SBS”), styrene/butadiene copolymer (such as“K-Resin®” available from Phillips 66 Co., Bartlesville, Okla.), etc.Preferably, the tube material is transparent. However, the tube does notnecessarily have to be clear, as long as the receiving instrument thatis looking for the cells or items of interest in the sample specimen can“see” or detect those items in the tube. For example, items of very lowlevel of radioactivity that can't be detected in a bulk sample, can bedetected through a non-clear or semi-transparent wall after it isseparated by the process of the present invention and trapped near thewall by the float 110 as described in more detail below.

In a preferred embodiment, the tube 130 is sized to accommodate thefloat 110 plus at least about five milliliters of blood or sample fluid,more preferably at least about eight milliliters of blood or fluid, andmost preferably at least about ten milliliters of blood or fluid. In anespecially preferred embodiment, the tube 130 has an inner diameter 138of about 1.5 cm and accommodates at least about ten milliliters of bloodin addition to the float 110.

The float 110 includes a main body portion 112 and two sealing rings orflanges 114, disposed at opposite axial ends of the float 110. The float110 is formed of one or more generally rigid organic or inorganicmaterials, preferably a rigid plastic material, such as polystyrene,acrylonitrile butadiene styrene (ABS) copolymers, aromaticpolycarbonates, aromatic polyesters, carboxymethylcellulose, ethylcellulose, ethylene vinyl acetate copolymers, nylon, polyacetals,polyacetates, polyacrylonitrile and other nitrile resins,polyacrylonitrile-vinyl chloride copolymer, polyamides, aromaticpolyamides (aramids), polyamide-imide, polyarylates, polyarylene oxides,polyarylene sulfides, polyarylsulfones, polybenzimidazole, polybutyleneterephthalate, polycarbonates, polyester, polyester imides, polyethersulfones, polyetherimides, polyetherketones, polyetheretherketones,polyethylene terephthalate, polyimides, polymethacrylate, polyolefins(e.g., polyethylene, polypropylene), polyallomers, polyoxadiazole,polyparaxylene, polyphenylene oxides (PPO), modified PPOs, polystyrene,polysulfone, fluorine containing polymer such aspolytetrafluoroethylene, polyurethane, polyvinyl acetate, polyvinylalcohol, polyvinyl halides such as polyvinyl chloride, polyvinylchloride-vinyl acetate copolymer, polyvinyl pyrrolidone, polyvinylidenechloride, specialty polymers, and so forth, and most preferablypolystyrene, polycarbonate, polypropylene, acrylonitritebutadiene-styrene copolymer (“ABS”) and others.

In this regard, one of the objectives of the present invention is toavoid the use of materials and/or additives that interfere with thedetection or scanning method. For example, if fluorescence is utilizedfor detection purposes, the material utilized to construct the float 110must not have much “background” fluorescence at the wavelength ofinterest.

The main body portion 112 and the sealing rings or support members 114of the float 110 are sized to have an outer diameter 118 which is lessthan the inner diameter 138 of the sample tube 130, under pressure orcentrifugation. The main body portion 112 of the float 110 is also lessthan the sealing or support rings 114, thereby defining an annularchannel or gap 150 between the float 110 and the sidewall 136 of thetube 130. The main body portion occupies much of the cross-sectionalarea of the tube, the annular gap 150 being large enough to contain thecellular components of the buffy coat layers and associated target cellswhen the tube is the non-flexed state. Preferably, the dimensions 118and 138 are such that the annular gap 150 has a radial thickness rangingfrom about 25-250 microns, most preferably about 50 microns.

A bore or channel 152 extends axially through the float 110. When thetube/float system is centrifuged, the tube expands, freeing the float inthe blood sample. As centrifugation is slowed, the float is captured bythe wall 136 of the tube as it returns to its original diameter. As thetube continues to contract, pressure may build up in the blood fractiontrapped below the float, primarily red blood cells. This pressure maycause red cells to be forced into the annular channel 150 containing thecaptured buffy coat layers, thus making imaging of the contents of thebuffy coat more difficult. Alternatively, the collapse of the side wallof the sample tube during deceleration may produce excessive ordisruptive fluid flow through the separated buffy coat layers. The bore152 allows for any excessive fluid flow or any resultant pressure in thedense fractions trapped below the float 110 to be relieved. Theexcessive fluid flows into the bore 152, thus preventing degradation ofthe buffy coat sample.

Although the depicted embodiments illustrate the preferred configurationof a central, axially-aligned bore 152, it will be recognized that otherconfigurations are contemplated so long as the bore extends completelyfrom one end to the other. In the preferred embodiment, the bore 152 iscentrally located and axially extending.

While in some instances the outer diameter 118 of the main body portion112 of the float 110 may be less than the inner diameter 138 of the tube130, this relationship is not required. This is because once the tube130 is centrifuged (or pressurized), the tube 130 expands and the float110 moves freely. Once the centrifugation (or pressurization) step iscompleted, the tube 130 constricts back down on the sealing rings orsupport ridges 114. The annular gap or channel 150 is then created, andsized by the height of the support ridges or sealing rings 114 (i.e.,the depth of the “pool” is equal to the height of the support ridges114, independent of what the tube diameter is/was).

In an especially preferred embodiment, the float dimensions are 3.5 cmtall×1.5 cm in diameter, with a main body portion sized to provide a50-micron gap for capturing the buffy coat layers of the blood. Thus,the volume available for the capture of the buffy coat layer isapproximately 0.08 milliliter. Since the entire buffy coat layer isgenerally less than about 0.5% of the total blood sample, the preferredfloat accommodates the entire quantity of buffy layer separated in aneight- to ten-milliliter sample of blood.

The sealing or support flanged ends 114 are sized to be roughly equalto, or slightly greater than, the inner diameter 138 of the tube. Thefloat 110, being generally rigid, can also provide support to theflexible tube wall 136. Furthermore, the large diameter portions 114provide a sealing function to maintain separation of the bloodconstituent layers. The seal formed between the large diameter regions114 of the float and the wall 136 of the tube may be, but is notnecessarily, a fluid-tight seal. As used herein, the term “seal” is alsointended to encompass near-zero clearance or slight interference betweenthe flanges 114 and the tube wall 136 providing a substantial seal,which is, in most cases, adequate for purposes of the invention.

The sealing rings 114 are most preferably continuous ridges, in whichcase the sample may be centrifuged at lower speeds and slumping of theseparated layers is inhibited. However, in alternative embodiments, theridges or sealing rings can be discontinuous or segmented bands havingone or openings providing a fluid path in and out of the annular gap150. The sealing rings or ridges 114 may be separately formed andattached to the main body portion 112. Preferably, however, the sealingridges 114 and the main body portion 112 form a unitary or integralstructure.

The overall specific gravity of the separator float 110 should bebetween that of red blood cells (approximately 1.090) and that of plasma(approximately 1.028). In a preferred embodiment, the specific gravityis in the range of from about 1.089-1.029, more preferably from about1.070 to about 1.040, and most preferably about 1.05.

The float may be formed of multiple materials having different specificgravities, so long as the composite specific gravity of the float iswithin the desired range. The overall specific gravity of the float 110and the volume of the annular gap 150 may be selected so that some redcells and/or plasma is retained within the annular gap, as well as thebuffy coat layers. Upon centrifuging, the float 110 occupies the sameaxial position as the buffy coat layers and target cells, e.g., thefloat 110 resting on the packed red cell layer. The buffy coat isretained in the narrow annular gap 150 between the float 110 and theinner wall 136 of the tube 130. The expanded buffy coat region can thenbe examined, e.g., under illumination and magnification, to identifycirculating epithelial cancer or tumor cells or other target analytes.

In one preferred embodiment, the density of the float 110 is selected toride in the granulocyte layer of the blood sample. The granulocytes ridein, or just above, the packed red-cell layer and have a specific gravityof about 1.08-1.09. In this preferred embodiment, the specific gravityof the float is in this range of from about 1.08 to about 1.09 suchthat, upon centrifugation, the float rides in the granulocyte layer. Theamount of granulocytes can vary from patient to patient by as much as afactor of about twenty. Therefore, selecting the float density such thatthe float rides in the granulocyte layer is especially advantageoussince loss of any of the lymphocyte/monocyte layer, which rides justabove the granulocyte layer, is avoided. During centrifugation, as thegranulocyte layer increases in size, the float rides higher in thegranulocytes and keeps the lymphocytes and monocytes at essentially thesame position with respect to the float.

The method for detecting circulating epithelial cancer or stem cells ina blood of a subject disclosed in U.S. Pat. No. 6,197,523 mayadvantageously be modified to employ the sample tube and float system ofthe subject invention. The aforementioned U.S. Pat. No. 6,197,523 isincorporated herein by reference in its entirety.

In a preferred exemplary method of using the tube/float system 100 ofthe invention, a sample of anticoagulated blood is provided. Forexample, the blood to be analyzed may be drawn using a standardVacutainer® or other like blood collection device of a type having ananticoagulant predisposed therein.

A fluorescently labeled antibody, which is specific to the targetepithelial cells or other analytes of interest, can be added to theblood sample and incubated. In an exemplary embodiment, the epithelialcells are labeled with anti-epcam having a fluorescent tag attached toit. Anti-epcam binds to an epithelial cell-specific site that is notexpected to be present in any other cell normally found in the bloodstream. A stain or colorant, such as acridine orange, may also be addedto the sample to cause the various cell types to assume differentialcoloration for ease of discerning the buffy coat layers underillumination and to highlight or clarify the morphology of epithelialcells during examination of the sample.

The blood is then transferred to the assembly 100 for centrifugation.The float 110 may be fitted into the tube 130 after the blood sample isintroduced into the sample tube 130 or otherwise may be placed thereinbeforehand. The tube and float assembly 100 containing the sample isthen centrifuged. Operations required for centrifuging the blood bymeans of the subject tube/float system 100 are not expressly differentfrom the conventional case, although, as stated above, reducedcentrifuge speeds may be possible and problems of slumping may bereduced. An adaptor may optionally be utilized in the rotor to preventfailure of the flexible tube due to stress.

When the centrifuging is started, the resultant hydrostatic pressuredeforms or flexes the wall 136 so as to enlarge the diameter of thetube. The blood components and the float 110 are thus free to move undercentrifugal motivation within the tube 130. The blood sample isseparated into six distinct layers according to density, which are, frombottom to top: packed red blood cells, reticulocytes, granulocytes,lymphocytes/monocytes, platelets, and plasma. The epithelial cellssought to be imaged tend to also collect in the buffy coat layers, i.e.,the granulocyte, lymphocyte/monocyte, and platelet layers as a result oftheir density. Due to the density of the float, it occupies the sameaxial position as the buffy coat layers and thus contents of the buffycoat occupy the narrow annular gap 150, potentially along with a smallamount of the red cell and/or plasma layers).

After centrifugal separation is complete and the centrifugal force isremoved, the tube 130 returns to its original diameter to capture orretain the buffy coat layers and other target analytes within theannular gap 150 for analysis. Optionally, the tube/float system 100 istransferred to a microscope or optical reader to identify any targetcells in the blood sample.

In one embodiment (see FIG. 3), the main body portion 312 has a diameterthat is smaller than the inner diameter of the tube and, thus, multipleannular channels 350 are defined between the main body portion 312 andthe inner tube wall. Optionally tapered ends 316 are provided tofacilitate and direct the flow of cells past the float 310 and sealingridges 314 during centrifugation. A central bore 352, shown in brokenlines, provides a pressure relief outlet to alleviate any pressure buildup in the lower fluid layers due to the contraction of the tube walls.Although the illustrated embodiment depicts continuous ribs, it will berecognized that the support ribs may likewise be broken or segmented toprovide an enhanced flow path between adjacent annular channels 350.

Referring now to FIG. 4, there is shown a splined separator float 410,including a plurality of axially oriented splines or ridges 424 whichare radially spaced about a central body portion 412. End sealing ridges414 and optionally tapered ends 416 are provided to facilitate anddirect the flow of cells past the float 410 and sealing ridges 414during centrifugation. The splines 424 and the end sealing ridges 414protrude from the main body 412 to engage and provide support for thedeformable tube once centrifugation is completed. The axial protrusions424 define fluid retention channels 450, between the tube inner wall andthe main body portion 412. The surfaces 413 of the main body portiondisposed between the protrusions 424 may be curved, e.g., when the mainbody portion 412 is cylindrical, however, flat surfaces 413 are alsocontemplated. Although the illustrated embodiment depicts splines 424that are continuous along the entire axial length of the float 410,segmented or discontinuous splines are also contemplated. A pressurerelief bore 452 extends axially and centrally through the float 410. Inother embodiments, one or more of such pressure relief bores, of similaror different shape, can be included in the main body of the float.

FIG. 5 illustrates a two-piece float 510 in accordance with a preferredembodiment of the present invention, shown in exploded view. A first,main body portion or sleeve 512 includes a central bore 552, which issized to slidably receive a second, piston-like center portion 554. Theouter body member 512 includes a flange or sealing ring 514, which is atits lower or bottom end. A sealing ridge or flange 515 is disposed atthe upper end of the piston section 554 during operation. Optionallytapered ends 517 are preferably provided at the upper and lower (duringoperation) ends of the piston portion 554 to facilitate and direct theflow of cells past the sealing ridges 514 and 515 during centrifugation.

The difference between the diameter of the main body 512 and thediameters of the sealing rings 514 and 515 are as described above by wayof reference to FIG. 1. In operation, the piston portion 554 is fullyreceived within the central bore 552 of the main body member 512. Asstated above, the float 510 is oriented in the tube so that the sealingridge 515 is at the top and the sealing ridge 514 is toward the bottomof the tube. The two portions may be formed of the same material ordifferent materials, so long as the overall specific gravity of thefloat 510 is in a suitable range for buffy coat capture. In anespecially preferred embodiment, the central piston portion 554 isformed of a slightly higher specific gravity material than the outerportion 512, which insures that the two portions stay together duringcentrifugation. Alternatively, the two float members are formed of thesame material and/or a frictional fit sufficient to keep the floatmembers together during centrifugation is provided.

As the tube containing the blood sample and float 510 is centrifuged,the two pieces 512 and 554 stay together and act in the same manner as aone-piece float to axially expand the buffy coat layers. When separationand layering of the blood components is complete and centrifugation isslowed, pressure may build in the red blood cell fraction trapped belowthe float, e.g., where contraction of the tube continues after initialcapture of the float by the tube wall. Any such pressure in the trappedred blood cell region forces the center piece 554 upward, thus relievingthe pressure, and thereby preventing the red blood cells from breechingthe seal between the sealing rings 514 and the tube wall.

FIGS. 6-12 illustrate further two-piece float embodiments of the presentinvention wherein the sealing rings are disposed at each end of theouter sleeve and pressure relief is provided by an upwardly movablepiston member.

FIG. 6 illustrates a two-piece float 610 including a first, main bodyportion or sleeve 612 having a central bore 652 slidably receiving asecond, piston-like center portion 654. The outer body member 612includes a sealing ring or ridge 614 at each end sized to engage thetube 130 (FIG. 1), with an annular recess 650 defined therebetween. Thepiston 654 includes a flanged end 656 that is greater in diameter thanthe central bore 652 and less than the diameter of the sealing ridges614.

In operation, the piston member 654 is fully received within the centralbore 652, with the flange 656 abutting the upper end of the sleeve 612.In use, the float 610 is oriented in the tube so that the flange 656 islocated toward the top of the tube 130, i.e., toward the stopper 140(FIG. 1). Again, the two portions may be formed of the same material ordifferent materials, so long as the overall specific gravity of thefloat 610 is in a suitable range for buffy coat capture. In anespecially preferred embodiment, the central portion 654 is formed of aslightly higher specific gravity material than the outer portion 612,which insures that the two portions stay together during centrifugation.Alternatively or additionally, a frictional fit is provided between thetwo float sections. Upon completion of centrifugation, any pressurebuild up in the trapped red blood cell region is alleviated by forcingthe center piece 654 upwardly.

FIG. 7 illustrates a two-piece float 710 similar to that shown anddescribed by way of reference to FIG. 6, but further including taperedends for facilitating blood flow around float 710 during centrifugation.A first, main body portion or sleeve 612 has a central bore 652 slidablyreceiving a second, piston-like center portion 754. The outer bodymember 612 includes sealing rings or ridges 614 at opposite ends, asdescribed above. The piston 754 includes a tapered end 756 including aflange 757 sized to abut the sleeve 612 upon insertion and restrict anyfurther downward passage of the piston 754. A lower end 758 of thepiston member 754 is also tapered to facilitate flow. Centrifugalmotivation and/or a frictional fit may be used to insure the twosections remain together during centrifugation.

FIG. 8 illustrates a two-piece float 810 including a first, main bodyportion or sleeve 812 having a central bore 852 and a counterbore 862,slidably receiving a second, piston-like center portion 854. The outerbody member 812 includes a sealing ring or ridge 814 as described above.The piston 854 includes a first, smaller diameter portion sized to bereceived within the central bore 852 and a second, larger diameterportion sized to be received within the counterbore 862. The axialextent of the small diameter segment 853 and large diameter segment 855may vary widely and are complimentary to that of the bore 852 andcounterbore 862, respectively.

Although the float 810 is shown with generally flat ends, it will berecognized that the ends of the piston member 854 and/or sleeve member812 may be tapered to facilitate fluid flow around the float duringcentrifugation. FIG. 9 illustrates an embodiment similar to that shownin FIG. 8, having tapered ends. A two-piece float 910 includes a first,main body portion or sleeve 912 having a central bore 952 and acounterbore 962, slidably receiving a second, piston-like center portion954. The outer body member 912 includes a sealing ring or ridge 914. Thepiston 954 includes a first, smaller diameter portion sized to bereceived within the central bore 952 and a second, larger diameterportion sized to be received within the counterbore 962. The taperedends 956 and 958 cooperate with complimentary end ridges to formgenerally conical ends.

Referring to FIGS. 8 and 9, during centrifugation, the float (810; 910)is oriented in the tube so that the counterbore and larger diameterportion are located toward the top of the tube 130 (FIG. 1). Asdescribed above, the two portions may be formed of the same material ordifferent materials and, in the preferred embodiment, the centralportion (854; 954) is formed of a slightly higher specific gravitymaterial than the outer sleeve (812; 912) insuring that the two sectionsstay together during centrifugation. Upon completion of centrifugation,any pressure built up in the trapped red blood cell region forces thecenter section (854; 954) upwardly.

FIG. 10 illustrates yet another two-piece float embodiment 1010including a first, main body portion or sleeve 1012 having a profiledbore comprising a central bore 1052 and an enlargement or countersink1062 opening toward the upper end of the tube. A second, piston-likemovable member 1054 includes a shaft 1053 and an enlarged head 1055,which are complimentary to and slidably received in the central bore1052 and the countersink 1062, respectively. The outer sleeve 1012includes sealing rings or ridges 1014 as described above. The float 1010is shown with tapered ends 1056 and 1058, however, it will be recognizedthat the ends of the float 1010 may also be flat. As described above,the two sections 1012 and 1054 may be formed of the same material ordifferent materials and, in the preferred embodiment, the movable member1054 is formed of a slightly higher specific gravity material than theouter sleeve 1012, insuring that the two sections stay together duringcentrifugation.

FIG. 11 illustrates a further two-piece separator float embodiment 1110including a first, main body portion or sleeve 1112 having a taperedinternal passage 1152 which widens toward the upper end 1156 of thefloat. A central, movable member 1154 complimentary to the bore 1152 isslidably received therein. The outer sleeve 1112 includes sealing ringsor ridges 1114. The separator float ends 1156 and 1158 are illustratedas tapered, although flat ends are also contemplated. The two sections1112 and 1154 may be formed of the same material or different materials,again, with the movable member 1154 preferably formed of a slightlyhigher specific gravity material to keep the float sections togetherduring centrifugation.

FIG. 12 illustrates a further two-piece separator float embodiment 1210including a first, main body portion or sleeve 1212 having an centralpassage or bore 1252 which terminates in an annular seat 1219 formed ata lower end of the float 1210 and defining an opening 1221 into the bore1252. A piston-like movable member 1254 is slidably received within thebore 1252, abutting the annular seat 1219. The outer sleeve 1212includes sealing rings or ridges 1214. The separator float 1210 isdepicted with flat ends, although tapered ends are also contemplated.Optionally, the movable member 1254 may contain a narrow diameterportion (not shown) on the lower end thereof sized to be received in theaperture 1221, e.g., to provide a flush and/or tapered surface tofacilitate flow therepast during centrifugation. The two sections 1112and 1254 may be formed of the same material or different materials;preferably, the movable member 1254 is formed of a slightly higherspecific gravity material to keep the float sections together duringcentrifugation.

Each of the float embodiments of FIGS. 1, 2, and 6-12, which have beenillustrated with end sealing rings and without additional tubesupporting members for ease of demonstration, may be further modified bythe further incorporation of any of the tube support features as shownand/or described in the above-incorporated U.S. patent application Ser.No. 10/263,975, now U.S. Pat. No. 7,074,577, such as annular bands,segmented bands, helical bands, axial splines, rounded protrusions,spikes, facets, and combinations thereof. Likewise, the separator floatembodiments are depicted herein having either flat or the preferredconical ends; however, many other geometrical shapes providing a curved,sloping, and/or tapered surface to facilitate density-motivated cell andfloat movement during centrifugation are contemplated, such as thoseshown and/or described in the above-incorporated U.S. patent applicationSer. No. 10/263,975, now U.S. Pat. No. 7,074,577. Exemplary modified endshapes include, for example, frustoconical, convex or dome-shaped, andother tapered shapes.

In use, a tube adapter may be used which insures sufficient expansion ofthe flexible tube to facilitate free movement of the separator floatwhile not allowing the tube to overexpand, which may lead to tubefailure, e.g., breakage or plastic deformation of the tube. Therefore, atubing adapter retaining the tube having an inner diameter sized toprovide a specific clearance gap between the adapter and the outerdiameter of the tube is advantageously used during centrifugation. Thisgap limits the expansion of the tube to a sufficient amount to allow thefloat to move freely in the tube, but not so much as to allow the tubeto fail.

Although suitable tubes are commercially available, an exemplary,preferred method for manufacturing the tubes addresses performanceattributes necessary to maintain and visualize the buffy coat. First, ithas been found that imperfections in the test tube provide wicking pathsfor red blood cells to intrude on the separated buffy coat retained inthe annular gap. Typically, the most severe imperfection found on thecommercially available test tubes is the molding parting line runningthe length of the test tube. Therefore, a sample tube was developed withthe parting line at the bottom of the tube where it does not interferewith the dynamics of the tube/float/blood interaction. Since thisprocess requires pulling the tubes from the injection mold rather thansplitting the mold, which produces the parting line, the top of thetest-tube may be thickened and flared out to facilitate using a stripperplate to remove the tubes. Other known methods for forming seamlesstubing may be employed as well.

In addition, the mold used consisted of a long core for the center ofthe sample tube. During molding, the pressure of the injection processtends to deform the core, producing uneven tube thicknesses. A featurefrom the main part of the mold was, therefore, added that mates with thefree-end of the core, the feature being the injector of the plastic. Themating action fixes the core to prevent deflection during injection.Late in the injection process, during injection, the feature iswithdrawn and plastic fills the remainder of the tube.

Efficient methods for manufacturing the floats have also been developed.In the case of the one-piece floats, injection molding is difficultbecause the thickness of the float makes it difficult to control theshrinkage of the plastic part, the amount of shrinkage beingproportional to the thickness of the part. Some of this concern isaddressed in the case of the two-piece floats by virtue of the fact twothinner parts that can be molded separately. That is, the thickness ofthese parts can be kept below about one-half inch, which typicallydefines the thickness limit for accurate molding. If further accuracy onany particular part, especially the outer dimension of the one-piece ortwo-piece floats, “over molding” can be employed. In this process, thepart is molded in two steps. The first step molds most of the partsmaller than desired, leaving a thin layer to be added later. Theshrinkage of the thin layer which is molded on in the second step can bemore precisely controlled, thus allowing a more precisely dimensionedpart to be molded.

Once the buffy layer is separated, it is desirable to present the tubeto an automated inspection system for imaging and analysis. Thisrequires precise positioning of the tube. Therefore, features may beadded to the sample tube, e.g., to the bottom of the tube, to facilitatetube engagement, handling, and positioning, e.g., under automated orpreprogrammed control.

The invention has been described with reference to the preferredembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations insofar as they come within thescope of the appended claims or the equivalents thereof.

1. A volume occupying separator float for use with an associated sampletube, comprising: a main body portion; a continuous, flat top supportmember protruding from and defining a top axial end of the main bodyportion; a continuous, flat bottom support member protruding from anddefining a bottom axial end of the main body portion; and an internalpassage extending axially completely through the main body portion,bottom support member, and top support member; wherein the main bodyportion has a diameter less than an inner diameter of the sample tube toform an annular volume when the float is within the sample tube.
 2. Thevolume occupying separator float of claim 1, wherein the float has aheight of 3.5 centimeters and a diameter of 1.5 centimeters.
 3. Thevolume occupying separator float of claim 1, wherein the float has aspecific gravity of from about 1.028 to about 1.090.
 4. The volumeoccupying separator float of claim 1, wherein the float has a specificgravity of from about 1.08 to about 1.09.
 5. The volume occupyingseparator float of claim 1, wherein the internal passage has a greatercross-sectional area at the top support member than at the bottomsupport member.
 6. A volume occupying separator float for use with anassociated sample tube for centrifugation, comprising: a rigid main bodyportion having a top axial end and a bottom axial end; a top sealingridge protruding radially from the top axial end of the main bodyportion; a bottom sealing ridge protruding radially from the bottomaxial end of the main body portion; and an internal passage extendingaxially completely through said main body portion; wherein the main bodyportion tapers from the top axial end towards the top sealing ridge andfrom the bottom axial end towards the bottom sealing ridge; and whereinthe top sealing ridge and the bottom sealing ridge protrude an equaldistance from the main body portion.
 7. The volume occupying separatorfloat of claim 6, wherein the float has a height of 3.5 centimeters anda diameter of 1.5 centimeters.
 8. The volume occupying separator floatof claim 6, wherein the float has a specific gravity of from about 1.028to about 1.090.
 9. The volume occupying separator float of claim 6,wherein the float has a specific gravity of from about 1.08 to about1.09.
 10. The volume occupying separator float of claim 6, wherein theinternal passage has a greater cross-sectional area at the top supportmember than at the bottom support member.
 11. The volume occupyingseparator float of claim 6, wherein the main body portion, top supportmember, and bottom support member form a unitary structure.
 12. Thevolume occupying separator float of claim 6, wherein the top and bottomsealing ridges both protrude from the main body portion to formcontinuous surfaces.
 13. The volume occupying separator float of claim6, wherein the main body portion tapers towards the top and bottomsealing ridges so as to form a curved surface at the top axial end andthe bottom axial end.
 14. The volume occupying separator float of claim6, wherein the main body portion tapers towards the top and bottomsealing ridges so as to form a sloped surface at the top axial end andthe bottom axial end.
 15. The volume occupying separator float of claim6, wherein the top axial end and the bottom axial end are conical. 16.The volume occupying separator float of claim 6, further comprising aplurality of ribs protruding radially from the main body portion, theribs being located between the top sealing ridge and the bottom sealingridge.
 17. The volume occupying separator float of claim 16, wherein theplurality of ribs protrude the same distance from the main body portionas the top and bottom sealing ridges.
 18. The volume occupying separatorfloat of claim 16, wherein the ribs are spaced equally apart.
 19. Thevolume occupying separator float of claim 16, wherein there are a totalof 8 ribs.